Biochemical and Biophysical Research Communications
Regular ArticleProduction and Characterization of Monoclonal Antibodies to N-Domain and Domain III of Carcinoembryonic Antigen
Abstract
In order to obtain MoAbs against N-domain or domain III of carcinoembryonic antigen (CEA), mice have been immunized with a recombinant deleted CEA which was devoid of most of domains I and II. Of the nineteen MoAbs established, ten MoAbs were reactive with the N-domain of CEA, and others recognized the domain III. All Fab fragments of the MoAbs against the N-domain significantly inhibited homophilic cell adhesion mediated by CEA, whereas that of normal mouse IgG or control MoAb (anti-HLA class II) did not. Among the Fab fragments of MoAbs against the domain III, three inhibited the cell adhesion slightly, while five enhanced and one had no effect. These findings suggest that the N-domain of CEA plays an important role in the cell adhesion, and that the domain III is also involved in the binding. The MoAbs described in this study will be useful to elucidate the functional roles of the domains of CEA molecule.
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Extracellular N-domain alone can mediate specific heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8
2000, Biochemical and Biophysical Research CommunicationsThe domain(s) responsible for the specific heterophilic adhesion between two members of the carcinoembryonic antigen (CEA) family, CEACAM6 and CEACAM8, both of which with three extracellular domains, were investigated using Chinese hamster ovary (CHO) transfectants expressing chimeric antigens. Using a chimeric antigen in which the N-domain, a sole extracellular domain, of CEACAM3 was substituted with that of CEACAM6, it was shown that the N-domain of CEACAM6 alone was able to mediate specific adhesion to CEACAM8. Furthermore, the chimeric antigen was shown to bind significantly to chimeric CEA whose N-domain was substituted with that of CEACAM8, but not to unsubstituted CEA. These results demonstrate that the N-domain alone is sufficient and other domains of CEACAM6 or CEACAM8 are not required for this specific binding. We therefore propose a model of heterophilic interaction between the N-domains, which is distinct from that of CEA–CEA homophilic binding.
Epitope mapping of monoclonal antibodies against N-domain of carcinoembryonic antigen
1999, Immunology LettersMonoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1–32, 1–32, and 33–59 of CEA, respectively, and that two discrete regions 1–32 and 60–93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27–29), eight residues (21, 27–29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA.
Humanization of a mouse neutralizing monoclonal antibody against tumor necrosis factor-α (TNF-α)
1999, Journal of Immunological MethodsAn anti-human tumor necrosis factor-α (TNF-α) monoclonal antibody, designated as 3B10, inhibits the biological activity of human TNF-α. In the present study, we constructed humanized version of the antibody by grafting its complementarity-determining regions (CDRs) onto a human antibody, HBS-1. Using a molecular model of mouse 3B10, framework residues affecting the CDR conformation were identified. Thus, these residues were also introduced into the framework together with the CDRs in a stepwise manner, depending on the degree of the possible importance of the residues. As a result, one humanized version (h3B10-9) which possesses nine mouse framework residues showed the same binding activity as that of the chimeric version. This humanized anti-TNF-α antibody is expected to be less immunogenic and thus more suitable for possible clinical use.
Homophilic intercellular adhesion mediated by C-CAM is due to a domain 1-domain 1 reciprocal binding
1996, Experimental Cell ResearchThe cell adhesion molecule C-CAM belongs to the immunoglobulin superfamily and is expressed in epithelia, vessel endothelia, and hematopoietic cells. Differential splicing gives rise to different isoforms, of which the major two are C-CAM1 and C-CAM2, which both have four Ig-like domains in their extracellular portions, but differ in their cytoplasmic domains. Two different allelic variants of C-CAM, namedaandb,occur in the rat. The adhesive binding mechanism(s) of C-CAM is not known in detail. Evidence for both homophilic and heterophilic binding has been presented, and different species and splice variants of C-CAM have shown differences in temperature and cation dependence when expressed in different cell types. Here, we have analyzed the binding mechanism of rat C-CAM2athat was expressed in CHO cells. In this system C-CAM2a-mediated adhesion was calcium- and temperature-independent. C-CAM2a-transfected cells did not adhere to nontransfected cells, demonstrating that the binding was homophilic. Cells transfected with C-CAM2ain which the N-terminal Ig-domain (D1) was deleted did not aggregate, and cells with intact C-CAM2acould not bind to these cells. This was in contrast to cells that were transfected with C-CAM2ain which the fourth Ig-like domain (D4) had been deleted; they both aggregated and bound to cells with intact C-CAM2a.Thus, C-CAM2amediates intercellular adhesion of CHO cells by a homophilic mechanism, in which the D1 domain binds reciprocally to a D1 domain on an opposed C-CAM molecule.
Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve fitting procedure: Implications for cellular adhesion
1996, Journal of Molecular BiologyCarcinoembryonic antigen (CEA) is one of the most widely used cell-surface tumour markers for tumour monitoring and for targeting by antibodies. It is heavily glycosylated (50% carbohydrate) and a monomer is constructed from one V-type and six C2-type fold domains of the immunoglobulin superfamily. The solution arrangement at low resolution of the seven domains in CEA cleaved from its membrane anchor was determined by X-ray and neutron scattering. Guinier analyses showed that the X-ray radius of gyrationRGof CEA was 8.0 nm. The length of CEA was 27 to 33 nm, and is consistent with an extended arrangement of seven domains. The X-ray cross-sectional radius of gyrationRXSwas 2.1 nm, and is consistent with extended carbohydrate structures in CEA. The neutron data gave CEA a relative molecular mass of 150,000, in agreement with a value of 152,500 from composition data, and validated the X-ray analyses. The CEA scattering curves were analysed using an automated computer modelling procedure based on the crystal structure of CD2. The V-type and C2-type domains in CD2 were separated, and the C2-type domain was duplicated five times to create a linear seven-domain starting model for CEA. A total of 28 complex-type oligosaccharide chains in extended conformations were added to this model. By fixing the six interdomain orientations to be the same, three-parameter searches of the rotational orientations between the seven domains gave 4056 possible CEA models. The best curve fits from these corresponded to a family of zig-zag models. The long axis of each domain was set at 160(±25)° relative to its neighbour, and the two perpendicular axes were orientated at 10(±30)° and −5(±35)°. Interestingly, the curve fit from this model is within error of that calculated from a CEA model generated directly from the CD2 crystal structure by the superposition of adjacent domains. Zig-zag models of this type imply that the protein face of the GFCC′ β-sheet in neighbouring CEA domains lie on alternate sides of the CEA structure. Such a model has implications for the adhesion interactions between CEA molecules on adjacent cells or for the antibody targeting of CEA.
Production and characterization of monoclonal antibodies against human natriuretic peptide receptor-A or -B
1995, Immunology LettersMonoclonal antibodies (mAbs) against human natriuretic peptide receptor-A (NPR-A) or NPR-B were produced using NPR-expressing Chinese hamster ovary (CHO) cells and soluble chimeric NPRs consisting of the extracellular domain of each receptor fused to Fc region of human IgG. Three anti-NPR-A mAbs, designated as A144, A397 and A416, bound to human NPR-A but not to NPR-B, while an anti-NPR-B mAb B136 reacted with human NPR-B but not with NPR-A. Competition analysis with the anti-NPR-A mAbs revealed that two mAbs, A144 and A416, recognize an identical or the adjacent site of the receptor and that A397 is directed against another epitope. No anti-NPR-A mAb affected binding of atrial natriuretic peptide (ANP) to NPR-A, while the anti-NPR-B mAb B136 inhibited binding of C-type natriuretic peptide (CNP) to NPR-B. Inhibition of the ligand-binding by B136 is specific in that the mAb showed no effect on the binding of ANP to NPR-A. B136 also blocked CNP-mediated intracellular cGMP accumulation in NPR-B-expressing cells. These results suggest that the region recognized by B136 may be related to the ligand-binding region of NPR-B. NPR-A- and NPR-B-expressing cells were selectively detected by immunostaining using the mAbs. These findings demonstrate that the mAbs will be useful to elucidate the role of the natriuretic peptides and their receptors in normal and disease states in human.